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Research article
Systematic identification of regulatory proteins critical for T-cell
activation
Peter Chu*
†
, Jorge Pardo*
†
, Haoran Zhao*
†
, Connie C Li*
‡
, Erlina Pali*,
Mary M Shen*, Kunbin Qu*, Simon X Yu*, Betty CB Huang*, Peiwen
Yu*
‡
, Esteban S Masuda*, Susan M Molineaux*, Frank Kolbinger
§
,
Gregorio Aversa
¶
, Jan de Vries
¶
, Donald G Payan* and X Charlene Liao*
#
Addresses: *Rigel Pharmaceuticals Inc., 1180 Veterans Blvd., South San Francisco, CA 94080, USA.
§
Novartis Pharma AG, S-386.6.25,
CH-4002 Basel, Switzerland.
¶
Novartis Forschungsinstitut GmbH, Brunner Strasse 59, A-1235 Vienna, Austria. Current addresses:
‡

Exelixis
Inc., 170 Harbor Way, South San Francisco, CA 94083, USA.
#
Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
†
These authors contributed equally to this work.
Correspondence: X Charlene Liao. E-mail: Donald G Payan. Email:
Abstract
Background: The activation of T cells, mediated by the T-cell receptor (TCR), activates a
battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling
proteins downstream of TCR activation will help us to understand the regulation of immune
responses and will contribute to developing therapeutic agents that target immune regulation.
Results: In an effort to identify novel signaling molecules specific for T-cell activation we
undertook a large-scale dominant effector genetic screen using retroviral technology. We
cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of
7×10
8
Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression
after expressing retrovirally derived cDNA libraries. We identified known signaling molecules
such as Lck, ZAP70, Syk, PLC␥1 and SHP-1 (PTP1C) as truncation mutants with dominant-
negative or constitutively active functions. We also discovered molecules not previously
known to have functions in this pathway, including a novel protein with a RING domain (found
in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1,
IL-10R␣ and integrin ␣
2
), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7,
SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using
truncated Lck, PLC␥1, EDG1 and PAK2 mutants as examples, we showed that these dominant
immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes.
Conclusions: This study identified important signal regulators in T-cell activation. It also

demonstrated a highly efficient strategy for discovering many components of signal transduction
pathways and validating them in physiological settings.
BioMed Central
Journal
of Biology
Journal of Biology 2003, 2:21
Open Access
Published: 15 September 2003
Journal of Biology 2003, 2:21
The electronic version of this article is the complete one and can be
found online at />Received: 19 August 2002
Revised: 3 July 2003
Accepted: 7 August 2003
© 2003 Chu et al., licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.
Background
Activation of specific signaling pathways in lymphocytes
determines the quality, magnitude and duration of immune
responses. These pathways are also responsible for the
induction, maintenance and exacerbation of physiological or
pathological lymphocyte responses in transplantation, acute
and chronic inflammatory diseases, and autoimmunity. The
activation of T lymphocytes is triggered when the T-cell
receptor (TCR) recognizes antigens presented by the major
histocompatibility complex (MHC) in antigen-presenting
cells [1]. Engagement of the TCR by antigen-MHC results in
rearrangement of the actin cytoskeleton, induction of gene
transcription, and progression into the cell cycle [2,3]. The
proximal events of TCR signaling include activation of the
Src-family kinases Lck and Fyn, phosphorylation of TCR

components, and activation of ZAP70 and Syk tyrosine
kinases, as well as recruitment of adaptor molecules (LAT
and SLP-76), which in turn couple to more distal signaling
pathways including Ras and PLC␥ [4,5]. Using classical
genetic and biochemical approaches, new components of
the TCR signaling pathway are being discovered, albeit at a
slow pace. Efficient identification of additional signaling
molecules probably requires novel approaches.
Here, we describe our attempt to identify and validate novel
signaling molecules specific for T-cell activation. We used
up-regulation of the cell-surface marker CD69 in T cells to
monitor TCR activation; CD69 as an activation marker has
been well validated [6], more recently using T cells deficient
in certain key signaling molecules such as SLP-76 and LAT
[7,8]. The rationale of this ‘functional genomics’ screen was
to identify cell clones whose CD69 upregulation was
repressed following introduction of clones from a retroviral
cDNA library. The library clones conferring such repression
would then represent immune modulators that function to
block TCR signal transduction.
Results
Experimental design
Jurkat Clone 4D9 was selected for low basal levels of CD69
expression and strong induction following TCR stimulation
(see Additional data file 1 with the online version of this
article for details of the selection and infection procedures).
The ‘Tet-off’ system was adapted for regulated expression of
the retroviral cDNA library: cDNA inserts in the retroviral
library were cloned behind the tetracycline (Tet) regulatory
element (TRE) and the cytomegalovirus (CMV) minimal

promoter. Transcription of the cDNA inserts was then
dependent on the presence of tetracycline-controlled trans-
activator (tTA) [9], a fusion of Tet repression protein and
the VP16 activation domain, and the absence of tetracycline
or its derivatives such as doxycycline (Dox). A derivative of
Jurkat clone 4D9 stably expressing tTA, called 4D9#32, was
engineered and selected (see Additional data file 1).
As a positive control for this functional genetic screen, we
tested dominant-negative forms of ZAP70, which are
known to inhibit TCR signaling [10]. We subcloned a
kinase-inactive ZAP70 (ZAP70 KI) and a truncated ZAP70,
comprising only the two Src homology 2 (SH2) domains
and referred to here as ZAP70 SH2 (N+C), into the bi-
cistronic retroviral vector under TRE control followed by the
internal ribosome entry site (IRES) coupled to green fluor-
escent protein (GFP; see Figure 1a). Both ZAP70 SH2 (N+C)
and ZAP70 KI inhibited TCR-induced CD69 expression
(Figure 1b). Consistent with previous reports using tran-
siently overexpressed ZAP70 constructs [10], the truncated
ZAP70 protein inhibited anti-TCR-induced CD69 expres-
sion more strongly than the ZAP70 KI protein did
(Figure 1b). The CD69-inhibitory phenotype was depen-
dent on expression of dominant-negative forms of ZAP70.
When Dox was added before TCR stimulation, there was no
inhibition of CD69 expression (Figure 1c, right panels). Flu-
orescence-activated cell sorting (FACS) analysis of cellular
expression of GFP revealed a lack of GFP-positive cells
(Figure 1c, left panels), suggesting that the bi-cistronic
ZAP70 SH2 (N+C)-IRES-GFP mRNA was not transcribed. A
lack of expression of the ZAP70 SH2 (N+C) protein in the

presence of Dox was confirmed by western blotting
(Figure 1d). Collectively, these results indicated that Jurkat
clone 4D9#32 was suitable for screening for inhibitors of
anti-TCR-induced CD69 expression.
Screening for cells lacking CD69 upregulation
The scheme to obtain cell clones with a CD69-inhibitory phe-
notype is shown in Figure 2a. Jurkat 4D9#32 cells were
infected with the pTRA-cDNA libraries made from human
lymphoid organs such as thymus, spleen, lymph node and
bone marrow (see Additional data file 2 with the online
version of this article for details of construction and assess-
ment of the pTRA-cDNA libraries). After library infection, cells
were stimulated with the anti-TCR antibody C305 overnight.
A total of 7.1 × 10
8
cells were stained with anti-CD69 anti-
body conjugated to allophycocyanin (APC) and anti-CD3
antibody conjugated to phycoerythrin (PE), and then screened
using flow cytometry. There was a significant reduction of the
CD3-TCR complex on the cell surface as compared to unstim-
ulated cells, as a result of receptor-mediated internalization,
but we were nevertheless able to distinguish the CD3
-
popu-
lation from the CD3
+
(CD3
low
and CD3
high

) populations (see
Additional data file 3 with the online version of this article
for the distinction between CD3
-
, CD3
low
and CD3
high
cell
populations). We consistently observed that more than 2% of
the cells had lost TCR-CD3 complex on the surface, causing
them to be unresponsive to stimulation and, consequently,
21.2 Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. />Journal of Biology 2003, 2:21
to have low CD69 expression (circled region R1 in
Figure 2b). We therefore collected by high-speed flow sorter
only cells with the lowest CD69 expression that still
retained CD3 expression. We termed the desired phenotype
CD69
low
CD3
+
(Figure 2a), and it represented 1% of the
total stained cells (boxed region R2 in Figure 2b). The 1%
sorting gate also translated as 100-fold enrichment in the
first round of sorting. In subsequent rounds of sorting, the
sorting gate R2 was always maintained to capture the equiv-
alent of 1% of the control cells that were stimulated but
were never flow-sorted. As shown in Figure 2b, we achieved
significant enrichment after three rounds of reiterative
Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. 21.3

Journal of Biology 2003, 2:21
Figure 1
Cell-line and assay development. (a) ZAP70 KI and ZAP70 SH2 (N+C) were subcloned in front of the internal ribosome entry site (IRES), followed
by GFP, in the Tet-regulated retroviral vector (pTRA-IRES-GFP). (b) After infecting tTA-expressing Jurkat (4D9#32) cells with retroviral constructs
containing IRES-GFP, ZAP70 KI-IRES-GFP, or ZAP70 SH2 (N+C)-IRES-GFP, cells were left unstimulated or stimulated with anti-TCR antibody for
24 h. CD69 expression was analyzed after gating on the GFP-positive population (infected population, boxed in R1). The dashed line and the thin line
on the graphs indicate cells infected with IRES-GFP (vector) before and after TCR stimulation, respectively, and the thick line indicates cells infected
with ZAP70 KI-IRES-GFP (top panel) or ZAP70 SH2 (N+C)-IRES-GFP (bottom panel), both after TCR stimulation. (c) After infecting Jurkat-tTA
(4D9#32) cells with retroviral vector alone or vector containing ZAP70 SH2 (N+C)-IRES-GFP, cells were cultured without (top panels) or with
(bottom panels) Dox for 6 days, and then left unstimulated or stimulated with anti-TCR antibody for 24 h. The box R1 indicates GFP-positive cells.
CD69 expression was analyzed for the entire cell population. The dashed line and the thin line indicate cells infected with vector before and after
TCR stimulation, respectively, and the thick line indicates cells infected with vector containing ZAP70 SH2 (N+C)-IRES-GFP after TCR stimulation.
(d) The Jurkat-tTA (4D9#32) cells containing different retroviral constructs (shown above the lanes) were cultured in the absence (-) or presence
(+) of Dox, and whole-cell lysates were prepared. Lysates were loaded (100 ␮g per lane) and analyzed by western blotting using anti-ZAP70
antibody (Upstate Biotechnology, Waltham, USA). The top ZAP70 band included endogenous (- and + Dox) as well as retrovirally expressed ZAP70
(-Dox only), whereas the bottom ZAP70 band contained only retrovirally expressed truncated ZAP70 SH2 (N+C).
SH2 SH2 Kinase
X
K369A
ZAP70
ZAP70 KI
ZAP70 SH2 (N+C)
Inactivated
LTR
GFPTRE
IRES
ZAP70 KI
Ψ
ZAP70 SH2 (N+C)
GFPTRE

IRES
R1
CD69
F
S
C
GFP
R1
10
0
10
1
10
2
10
3
10
4
400
320
240
160
80
0
10
0
10
1
10
2

10
3
10
4
400
320
240
160
80
0
10
0
10
1
10
2
10
3
10
4
1000
800
600
400
200
0
Cells in R1
Cells in R1
10
0

10
1
10
2
10
3
10
4
1000
800
600
400
200
0
Vector – anti-TCR
Vector + anti-TCR
ZAP70 SH2 (N+C)
+ anti-TCR
Vector − anti-TCR
Vector + anti-TCR
ZAP70 KI + anti-TCR
Events
Events
R1
All cells
+ Dox
GFP
F
S
C

CD69
− Dox
10
0
10
1
10
2
10
3
10
4
500
400
300
200
100
0
10
0
10
1
10
2
10
3
10
4
500
400

300
200
100
0
10
0
10
1
10
2
10
3
10
4
1000
800
600
400
200
0
10
0
10
1
10
2
10
3
10
4

1000
800
600
400
200
0
R1
Vector − anti-TCR
Vector + anti-TCR
ZAP70 SH2 (N+C)
+ anti-TCR
− Dox
+ Dox
Events
Events
All cells
ZAP70 SH2
(N+C)
ZAP70
Jurkat
4D9#32 Vector
ZAP70 ZAP70 KI
ZAP70 SH2 (N+C)
Dox
− + − + − + − +
64 –
51 –
39 –
28 –
19 –

14 –
M
r
(kDa)
Inactivated
LTR
Inactivated
LTR
Inactivated
LTR
(a)
(d)
(b)
(c)
sorting; cells with the desired CD69
low
CD3
+
phenotype
increased from 1% to 23.2% of the population. In addition,
the overall population’s geometric mean for the CD69
fluorescent intensity was also reduced (from > 300 to 65).
Given our experimental design, we expected the expression
of retroviral cDNAs and their putative inhibitory effect to be
turned off with the addition of Dox. This feature helped us
to ascertain that the phenotype was due to expression of the
cDNA library rather than to epigenetic changes or sponta-
neous or retroviral-insertion-mediated somatic mutation(s).
To confirm this, we compared anti-TCR-induced CD69
expression in the presence and absence of Dox. As shown in

Figure 2c, cells with the CD69
low
CD3
+
phenotype decreased
from 24.0% to 13.0% with the addition of Dox, demon-
strating that a significant number of cells (11%) had lost the
CD69
low
CD3
+
phenotype when library-cDNA expression
was turned off. These data suggested that the CD69
low
CD3
+
phenotype in a significant proportion (at least 11% out of
24%, or 45.8%) of cells in this population was indeed
caused by expression of the cDNA-library clones.
Functional analysis of single-cell clones
Next, we deposited single cells into 96-well plates in con-
junction with the fourth and subsequent rounds of sorting
for the CD69
low
CD3
+
phenotype. The phenotype of each
single-cell clone was characterized by growing the cells in
the absence and presence of Dox. A few examples of the
Dox-regulatable phenotypes for individual clones are

shown in Figure 3a. Dox regulation of CD69 expression was
expressed as the ratio of CD69 geometric mean fluorescent
intensity in the presence of Dox divided by the CD69 geo-
metric mean fluorescent intensity in the absence of Dox
after TCR stimulation; we termed this ratio the ‘Dox ratio’.
In uninfected or mock-infected cells, Dox had little or no
effect on the induction of CD69 expression, with mean Dox
21.4 Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. />Journal of Biology 2003, 2:21
Transfect Phoenix cells with pTRA-cDNA libraries
(total complexity of 5 x 10
7
)
Collect viral supernatant
Activate with anti-TCR
Sort CD69
low
CD3
+
cells
Single cells cloned into 96-well plates
Repeat
Functional analysis of single-cell clones (± Dox)
RT-PCR cloning of cDNA inserts
Infect 3.5 x10
8
Jurkat-tTA 4D9 #32
R2
R2
R2
R2

CD3
No sort
After three rounds
1.1%
1.0% 23.2%
2.6%
Y Geo Mean = 316
Y Geo Mean = 340
Y Geo Mean = 65
Y Geo Mean = 291
CD69
10
4
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
10

4
10
4
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
10
4
10
4
10
3
10
2
10
1
10

0
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
4
10
3
10
2
10
1
10

0
10
0
10
1
10
2
10
3
10
4
10
4
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
10

4
10
4
10
3
10
2
10
1
10
0
10
0
10
1
10
2
10
3
10
4
After two rounds
After one round
R1
R2R2
CD3
− Dox + Dox
24.0% 13.0%
Y Geo Mean
= 68

CD69
− Dox
+ Dox
CD69
Y Geo Mean
= 106
200
160
120
80
40
0
Events
(a)
(b)
(c)
Figure 2
Screen for inhibitors of TCR-activation-induced CD69 expression.
(a) Cells (3.5 × 10
8
) were infected with pTRA-cDNA libraries. Single-
cells were cloned after at least four consecutive sortings of the
CD69
low
CD3
+
phenotype. (b) Cells (7.1 × 10
8
) were sorted with high-
speed flow sorters (MoFlo) after stimulation and staining with anti-

CD69-APC and anti-CD3-PE. The sort gate was set at the equivalent of
1% of satellite control cells that were stimulated but never flow-sorted
(shown as R2) to enrich for the CD69
low
CD3
+
phenotype. After
sorting, the desired cells were allowed to rest for 6 days before
another round of stimulation and sorting. (c) Cells were split into two
populations after the third round of sorting. One half of the cells were
grown in the absence of Dox (top left dot-plot) and the other half in
the presence of Dox (top right dot-plot). Six days later, CD69
expression was compared following anti-TCR stimulation. The dashed
line indicates CD69 level without Dox and the solid line with Dox
(bottom graph).
ratios for individual clones of 1.00 ± 0.25 (standard devia-
tion). We used twice the standard deviation above the mean
as a cut-off criterion and regarded clones with a ratio above
1.5 as Dox-regulated clones. Out of 2,828 clones analyzed,
1,323 had a Dox-regulatable phenotype, representing
46.8% of analyzed clones. This percentage was comparable
to the percentage based on the overall population (46.8%
compared to 45.8%), suggesting that the single-cell clones
constituted a fair representation of the entire population.
The distribution of Dox ratios among all 2,828 clones is
shown in Additional data file 4, with the online version of
this article.
The cDNA inserts of selected clones with a Dox-regulatable
phenotype were recovered by RT-PCR using primers specific
for the vector sequence flanking the cDNA library insert

(Figure 3b). Most clones generated only one RT-PCR
product, but a few clones generated two or more products.
Sequencing analysis revealed that the additional RT-PCR
products were usually caused by double or multiple inser-
tions of retroviruses. The results of the cDNA analysis are
summarized in Table 1.
Characterization of proteins critical for T-cell
activation
As shown in Table 1, we obtained known TCR regulators
such as Lck, ZAP70, Syk, PLC␥1, PAG, SHP-1/PTP1C, Csk
and nucleolin (reviewed in [11]). The hits with the highest
frequency, however, were those encoding the TCR ␤ subunit.
This new ␤ chain leads to the assembly of a new TCR
complex no longer recognizable by the stimulating antibody
C305, because C305 only recognizes the original endoge-
nous Jurkat clonotypic TCR complex [2] (see also Additional
data file 5, with the online version of this article).
Among the known T-cell activation regulators, we obtained
two ZAP70 hits containing the endogenous ATG initiation
codon, missing the catalytic domain and ending at amino
acids 262 and 269, respectively (Figure 4a). The deletions
closely mirror the positive control for the screen, ZAP70
SH2 (N+C), which ended at amino acid 276 and has been
shown to be a dominant-negative protein [10]. Similarly,
we obtained a kinase-truncated form of Lck (Figure 4b) that
caused inhibition of CD69, mimicking the phenotype of a
Jurkat somatic mutant lacking Lck [12]. These clones repre-
sent dominant-negative forms of kinases required for T-cell
activation. The inhibitory effects of these and other clones
were confirmed by subcloning them into the pTRA-IRES-

GFP vector, reintroducing into the naïve Jurkat-tTA cells,
and comparing the CD69 expression in GFP-positive and
GFP-negative cells upon TCR stimulation (Figure 4).
TCR engagement leads to rapid tyrosine phosphorylation
and activation of PLC␥1 [13]. One of our hits contained
the pleckstrin homology (PH) domain and the amino-
and carboxy-terminal SH2 domains of PLC␥1 (Figure 4c).
Significantly, this hit also lacked the crucial tyrosine Y783,
which is essential for coupling of TCR stimulation to IL-2
promoter activation. The Y783F mutant is a very potent
dominant-negative form of PLC␥1 [14]. Indeed, the origi-
nal clone encoding the PLC␥1 hit had the highest Dox
ratio for CD69 expression among all clones analyzed.
Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. 21.5
Journal of Biology 2003, 2:21
Figure 3
Identification of clones with desired phenotype. (a) Individual clones
were grown in the presence (open peaks) or absence (filled peaks) of
Dox for 6 days and then stimulated to examine CD69 expression by
FACS. The ‘Dox ratio’ was defined as the ratio of CD69 geometric
mean fluorescent intensity in the presence of Dox divided by CD69
geometric mean fluorescent intensity in the absence of Dox and is
indicated in parentheses following the clone number. (b) DNA
oligonucleotide primers specific to the library vector (BstXTRA5G and
BstXTRA3D, not to scale) were used in RT-PCRs to recover the
cDNA inserts from cell clones. The RT-PCR products were analyzed in
agarose gel followed by ethidium blue staining. Data from
representative clones are shown alongside the 1kb DNA molecular
weight ladder (M
r

) from New England BioLabs (Beverly, USA).
Clone 15 (17.15)
Clone 24 (12.43)
Clone 64 (13.80)
Clone 116 (5.27) Clone 157 (69.90) Clone 194 (9.30)
20
15
10
5
0
20
15
10
10
0
10
1
10
2
10
3
10
4
5
0
20
15
10
10
0

10
1
10
2
10
3
10
4
5
0
20
15
10
10
0
10
1
10
2
10
3
10
4
5
0
10
0
10
1
10

2
10
3
10
4
20
15
10
10
0
10
1
10
2
10
3
10
4
5
0
20
15
10
10
0
10
1
10
2
10

3
10
4
5
0
Events Events
CD69
∆U3 R U5∆U3 R U5
TRE/Pmin
SD SA SA SD
cDNA
SD SA
BstXTRA3D
cDNA insert
BstXTRA5G
pA
5′ 3′
Ψ
pA
(a)
(b)
21.6 Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. />Journal of Biology 2003, 2:21
Table 1
Overview of identified molecular targets
Accession Phenotype
Gene Domain homology Direction number* Relative to ORF* Frequency* transfer
Known to function in TCR pathway
TCR␤ Receptor Sense Numerous Partial 46 On hold
ZAP70 Tyrosine kinase Sense L05148.1 -147, +787 nt 12 Yes
ZAP70 (long) Tyrosine kinase Sense L05148.1 -21, +809 nt 17 TBD

Syk Tyrosine kinase Sense L28824.1 -27, +1012 nt 2 Yes
Lck Tyrosine kinase Sense U23852.1 -59, +799 nt 4 Yes
PLC␥1 Tyrosine kinase Sense NM_002660.1 +1409, +2282 nt 3 Yes
SHP-1/PTP1C Protein-tyrosine Sense X62055.1 +472, >+2021 nt 1 Yes
phosphatase
Csk Tyrosine kinase Sense NM_004383.1 -55, +1285 nt 1 TBD
PAG Transmembrane adaptor Sense NM_018440.2 -237, +644 nt 1 Yes
Nucleolin RNA-binding Sense NM_005381.1 -136, +479 nt 1 No
Enzymes and receptors
TCPTP/PTPN2 Protein-tyrosine phosphatase Sense NM_002828.1 -58, +1108 nt 20 Yes
PAK2 p21-activated kinase 2 Sense NM_002577.1 -50, +339 nt 18 Yes
PAK2 (long) p21-activated kinase 2 Sense NM_002577.1 -42, +670 nt 1 Yes
A-Raf-1 Serine/threonine kinase Sense X04790.1 -4, +456 nt 5 Yes
EDG1 G-protein-coupled receptor Sense NM_001400.2 <-244, +942 nt 4 Yes
EDG1 (long) G-protein-coupled receptor Sense NM_001400.2 <-244, +1037 nt 1 TBD
TRAC-1 RING finger ubiquitin ligase Sense NM_017831.1 -254, +510 nt 1 Yes
IL-10R␣ Receptor Sense NM_001558.1 +689, +1350 nt 1 Yes
Integrin ␣
2
Receptor Sense NM_002203.2 +3348, +3914 nt 1 Yes
Enolase 1␣ Phosphopyruvate hydratase Sense NM_001428.1 +703, +1374 nt 2 No
DUSP1 Dual-specificity phosphatase Sense NM_004417.2 +817, +1112 nt 1 No
KIAA0251 Pyridoxal-dependent Sense D87438.1 nt 2098-2370
†
1No
decarboxylase
Adaptors and transcription factors
Grb7 Adaptor Sense NM_005310.1 +1268, +1912 nt 3 Yes
GG2-1 TNF-induced protein Sense AF070671.1 -97, +1795 nt 2 Yes
SH2-B Adaptor Sense AF227968.1 +1352, +1960 nt 1 Yes

RERE Transcriptional factor Sense AB036737.1 +914, +1202 nt 3 No
SudD Serine/threonine rich Sense NM_003831.1 -93, +413 nt 1 No
Ku 70 DNA-PKc subunit Sense S38729.1 +1026, +2069 nt 1 No
Novel (130 Unknown Sense AC005321.1 nt 33543-33938
‡
1No
amino acids;
no homology)
Novel signaling GYF domain Sense NM_022574.1 +1, +121 nt 1 No
molecule
SCAMP2 Secretory carrier Sense AF005038.2 -5, +833 nt 1 No
membrane protein
KIAA1228 C2 domain (Ca
2+
- or Sense AB033054.2 nt 1439-2163
†
1No
IP-binding)
EST from LPP20 lipoprotein precursor Sense Al357532.1 Novel isoform 1 No
clone 2108068
RNH Ribonuclease/angiogenin Sense NM_002939.1 -22, +713 nt 1 No
inhibitor
When introduced into naïve Jurkat cells, this fragment also
caused a severe block of TCR-induced CD69 expression
(Figure 4c).
In addition to known signaling molecules, we also discov-
ered genes whose sequences had been reported previously
but whose involvement in TCR signaling was not docu-
mented (Table 1, and see Additional data files 6 and 7 with
the online version of this article). EDG1 (endothelial differ-

entiation gene-1) was discovered initially from a set of
immediate-early-response gene products cloned from
human umbilical vein endothelial cells [15]. EDG1 is a G-
protein-coupled receptor (GPCR) with high affinity for
sphingosine 1-phosphate (S1P) [16]. Although EDG1 has
been reported to link to multiple signaling pathways [17],
no role in TCR signaling had been documented. From our
genetic screen, we obtained two carboxy-terminal trunca-
tion EDG1 mutants. Reintroducing EDG1 Hit 1 into naïve
Jurkat cells conferred a CD69-inhibition phenotype
(Figure 4d). We believe the EDG1 hits may work as consti-
tutively active forms of the endogenous protein, given that
overexpressing full-length EDG1 also caused inhibition of
CD69 expression (data not shown).
PAK (p21-activated kinase) proteins are critical effectors
that link Rho-family GTPases, such as Cdc42 and Rac1, to
cytoskeletal reorganization and nuclear signaling [18,19].
PAK proteins constitute a family of serine/threonine kinases
that utilizes the CRIB (Cdc42/Rac interactive binding)
domain to bind to small GTPases; members of the family
include PAK1, PAK2, PAK3 and PAK4 [19]. Among the four
PAK proteins, PAK2 (also known as PAK65 [20] and
gamma-PAK [21]) is activated by proteolytic cleavage
during caspase-mediated apoptosis [22]. The role of PAK2
in Jurkat T cells has been reported primarily to be in mem-
brane and morphological changes in apoptotic cells [23].
PAK1, on the other hand, has been reported to be involved
in T-cell signaling [24,25]. Interestingly, we identified two
different truncated versions of PAK2, both lacking the
kinase domain, in our functional genetic screens with the

fourth highest frequency (after TCR␤, ZAP70 and TCPTP;
see Table 1). We further demonstrated that these dominant-
negative forms of PAK2 also confer CD69 inhibition when
introduced into naïve Jurkat cells (Figure 4e and Table 1).
An interesting adaptor molecule cloned from our genetic
screen is Grb7 (Figure 4f). Like Grb2, Grb7 was originally
cloned by screening bacterial expression libraries with the
tyrosine-phosphorylated carboxyl terminus of the epider-
mal growth factor (EGF) receptor [26]. The Grb7 family of
proteins - Grb7, Grb10, and Grb14 - share significant
sequence homology and a conserved molecular architecture
[27]. Their functional domains include a proline-rich
region, an RA (RalGEF/AF6 or Ras-associating) domain, a
PH domain and an SH2 domain. Like other adaptor mole-
cules, Grb7 family proteins function to mediate the cou-
pling of multiple cell-surface receptors to downstream
signaling pathways in the regulation of various cellular
functions. Our identification of a strong phenotype for the
Grb7 SH2 domain in TCR signal transduction suggests that
Grb7 may be an important immune-regulatory molecule
(Figure 4f).
Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. 21.7
Journal of Biology 2003, 2:21
Table 1 (continued)
Overview of identified molecular targets
Accession Phenotype
Gene Domain homology Direction number* Relative to ORF* Frequency* transfer
Cytoskeleton
Moesin Moesin Sense NM_002444.1 -93, +1534 nt 1 Yes
Vimentin Intermediate filament Sense NM_003380.1 -98, +374 nt 1 Yes

Others
Alu repeat 5 On hold
CpG island? AL035420.1 1 On hold
(clone 550H1)
IgG2 heavy chain Ig superfamily Sense Partial 1 On hold
Ig light chain Ig superfamily Sense Partial 1 On hold
18S rRNA Sense M10098.1 Partial 2 On hold
28S rRNA Sense Partial 1 On hold
*For each identified clone, the GenBank database [51 ] accession number is given, followed by the first and last nucleotide (nt) positions relative to
the initiation codon (ATG being the +1, +2, +3 nts, respectively); Frequency indicates the number of original cell clones expressing the specific hit.
†
Relative to the EST itself because the start codon is not identified.
‡
Relative to the genomic clone itself. Ig, Immunoglobulin; TBD, to be determined.
21.8 Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. />Journal of Biology 2003, 2:21
Figure 4 (see the legend on the next page)
ZAP70
Hit 1
1
619
262
SH2
1
Protein kinaseSH2
SH2 SH2
Hit 2 (long)
269
1
SH2 SH2
CD69

Original clone for hit 1
CD69
Dox ratio = 7.98
Events
EventsEvents
GFP−
GFP+
− Anti-TCR
+ Anti-TCR
Lck
Hit
1
363
266
SH3
1
Protein kinase
SH2
SH3 SH2
GFP−
GFP+
CD69
Original clone
CD69
Dox ratio = 2.9
Events
EventsEvents
− Anti-TCR
+ Anti-TCR
PLCγ1

Hit
1
1290
761
470
PLC-YPLC-XPH
SH2 SH2 SH3
C2
SH2 SH2
PH
PH
PH
CD69
Original clone
CD69
Dox ratio = 71.7
Events
EventsEvents
GFP−
GFP+
− Anti-TCR
+ Anti-TCR
EDG1
1 381
Seven-transmembrane
1 314
Seven-transmembrane
1
345
Seven-transmembrane

49 313
Hit 1
Hit 2 (long)
CD69
Original clone for hit 1
CD69
Dox ratio = 8.7
Events
Events
GFP−
− Anti-TCR
+ Anti-TCR
Events
GFP+
1
524
113
CRIB
1
Protein kinase
1 224
249
PAK2
Hit 1
Hit 2 (long)
Dox ratio = 12.5
Original clone for hit 1
CD69
Events
Events

CD69
Events
GFP−
− Anti-TCR
+ Anti-TCR
GFP+
CD69
Original clone
CD69
Grb7
Hit
1 532
532
422
SH2
RA
100 186 230 338 431 512
SH2
SH2
PHPro BPS
GFP−
− Anti-TCR
+ Anti-TCR
Events
Events
GFP+
Dox ratio = 7.9
Events
TRAC-1
Hit

1
232 aa
170 aa
1
RING
RING
37 75
37 75
Dox ratio = 7.3
CD69
Events
Events
CD69
Events
Original clone
GFP−
− Anti-TCR
+ Anti-TCR
GFP+
30
25
10
5
10
0
10
1
10
2
10

3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10

3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10

3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10

3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10

3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10

3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10

3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
0
20
15
150
120
90
60

30
0
60
50
20
10
0
40
30
50
40
30
20
10
0
500
400
300
200
100
0
50
40
30
20
10
0
30
25
10

5
0
20
15
100
80
60
40
20
0
0
20
10
40
30
30
25
10
5
0
20
15
400
320
240
160
80
0
0
10

5
20
15
0
10
5
20
15
500
400
300
200
100
0
50
40
30
20
10
0
10
5
0
20
15
800
640
480
320
160

0
100
80
60
40
20
0
30
25
10
5
0
20
15
500
400
300
200
100
0
120
100
40
20
0
80
60
(a) (b)
(c) (d)
(e)

(g)
(f)
We also discovered an uncharacterized molecule whose
sequence in GenBank was assembled from expressed
sequence tag (EST) data. This novel molecule, FLJ20456, was
renamed by us as TRAC-1, for T-cell RING protein in activa-
tion. As shown in Figure 4g, TRAC-1 has a RING finger
domain, which is characteristically found in a class of pro-
teins collectively called ubiquitin ligases or E3s [28].
Members of the Cbl protein family are the best-known E3s
involved in the regulation of TCR signaling [29]. T cells mani-
fest enhanced signaling in both c-Cbl and Cbl-b mutant mice,
suggesting that the wild-type function of these proteins is in
negatively regulating T-cell activation. More recently, Cbl pro-
teins have been shown to function as RING finger E3s so as
specifically to target activated receptors and protein-tyrosine
kinases for ubiquitination and therefore to down-regulate
their signaling [30]. The TRAC-1 hit we obtained has a trunca-
tion in the carboxyl terminus but still retains the intact RING
finger domain (Figure 4g). Reintroducing the TRAC-1 hit into
naïve Jurkat cells caused strong inhibition of the anti-TCR-
induced CD69 expression in infected cells.
For a complete characterization of the functional genetic
screen, as well as additional selected hits, see Additional
data files 6 and 7 with the online version of this article.
Gene expression in tissues and primary lymphocytes
We studied the expression profiles of EDG1, PAK2, Grb7
and TRAC-1 by northern blot analysis. We detected ubiqui-
tous expression of EDG1 and PAK2 in normal human
tissues, including thymus, spleen and peripheral blood lym-

phocytes (PBL; Figure 5a). Grb7 has strong expression in
kidney and placenta, but little or no expression in thymus
or PBL by northern blot analysis (Figure 5b). Interestingly,
TRAC-1 has a highly specific expression in organs associated
with the lymphoid system or hematopoietic system, such as
spleen, liver and PBL (Figure 5b). We also detected a faster-
migrating band with the TRAC-1 probe in placenta, perhaps
representing an alternatively spliced message.
We further examined expression of these selected genes in
lymphocyte subsets isolated from healthy human peripheral
blood using semi-quantitative RT-PCR. As shown in
Figure 5c, EDG1 expression was detected in both T cells
(higher expression in CD4
+
than in CD8
+
T cells) and B cells
(CD19
+
), but not in monocytes (CD14
+
). Its expression
level in T and B cells was not affected upon mitogenic acti-
vation. EDG1 was also detected in the brain. PAK2 was
detected in resting and activated lymphocytes as well as in
the placenta (Figure 5d). Even though Grb7 was not
detected in the PBL by northern blot, it was detected in
peripheral blood mononuclear cells (PBMC) using the more
sensitive RT-PCR method (Figure 5e). Grb7 expression
seemed to be slightly increased upon activation. Consistent

with the northern blot profile, TRAC-1 was detected only in
lymphocytes and not in the placenta (Figure 5f). In
summary, all four genes are expressed in the lymphoid
system, supporting their potential physiological role in lym-
phocyte signaling.
Function in primary T lymphocytes
The relevance of the cDNA hits from our screen to the physio-
logical functions of T cells was investigated in primary T lym-
phocytes. We subcloned the hits into a retroviral vector under
the control of a constitutively active promoter embedded in
the retroviral long terminal repeat (LTR), followed by IRES-
GFP [31]. We then developed a protocol to couple successful
retroviral infection to subsequence T-cell activation. As
shown in Figure 6a (left panels), fresh PBL contained both T
cells and B cells. The combined CD4
+
and CD8
+
cells repre-
sented T cells (about 81% of total lymphocytes in this partic-
ular donor). The remaining 19%, which were CD4
-
CD8
-
cells,
were B cells as stained by CD19 (data not shown). Upon cul-
turing with anti-CD3 and anti-CD28 antibodies, primary T
lymphocytes were expanded and primary B cells and other
cell types gradually died off (Figure 6a, right panels). Impor-
tantly, primary T lymphocytes were successfully infected by

retroviruses (Figure 6a,b).
As seen with Jurkat cells (data not shown), GFP translated
by way of IRES was not as abundant as GFP translated using
the conventional Kozak sequence (comparing GFP geomet-
ric mean from CRU5-IRES-GFP to that from CRU5-GFP).
Nevertheless, the percentage infection remained similar
(Figure 6b; 32.4% and 31.3% respectively). Insertion of a
gene in front of IRES-GFP further reduced the expression
level of GFP (Figure 6b), a trend observed with many other
Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. 21.9
Journal of Biology 2003, 2:21
Figure 4 (see the figure on the previous page)
Transfer of selected hits from the functional genetic screen to naïve Jurkat-tTA (4D9#32) cells. Diagrams of proteins predicted from the cDNA
inserts and those from the corresponding wild-type genes are shown above the histograms. The left panel of histograms shows the phenotype of the
original cell clones in the presence (open peaks) or absence (filled peaks) of Dox as analyzed in Figure 3a. The Dox ratio is indicated. The right top
and bottom panels of histograms show the phenotypes after expressing the cDNA inserts (followed by IRES-GFP) in a naïve Jurkat-tTA population.
After retroviral infection, the Jurkat-tTA (4D9#32) cells were either stimulated with the anti-TCR antibody (solid line) or left unstimulated (dashed
line), and analyzed by FACS for CD69 induction after staining with anti-CD69-APC. The top right histogram in each group analyzed GFP-negative
cells, which did not express the cDNA hit, whereas the bottom right histogram in each group analyzed GFP-positive cells, which expressed the
cDNA hit. The following cDNA hits are shown: (a) ZAP70; (b) Lck; (c) PLC␥1; (d) EDG1; (e) PAK2; (f) Grb7; (g) TRAC-1.
cell lines (data not shown). After allowing cells to rest for
5 days following infection, we flow-sorted cells into two
populations: GFP-negative and GFP-positive. Exact numbers
of sorted cells were immediately put into culture. As seen in
Figure 6c, resting cells did not produce IL-2, nor did cells
stimulated with anti-CD3 alone. Anti-CD3 plus anti-CD28
induced robust IL-2 production in the CIG vector-infected
cells (CIG), regardless of the GFP expression (note the differ-
ent scales of the upper graphs compared to the lower ones).
These observations are consistent with previous reports on

freshly isolated primary T lymphocytes and also indicate
21.10 Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. />Journal of Biology 2003, 2:21
Figure 5
EDG1, PAK2, Grb7 and TRAC-1 expression in normal human tissues and lymphocyte subsets. (a,b) Northern blot analysis using multi-tissue blot
(Clontech). The following genes are shown: (a) EDG1 and PAK2; (b) Grb7 and TRAC-1. (c-f) Semi-quantitative PCR analysis of gene expression in
lymphocyte subsets. The cDNA templates were obtained from CD4
+
T cells, CD8
+
T cells, CD19
+
B cells, or CD14
+
monocytes (human blood
fractions MTC panel from Clontech). Specific target primers or control primers were used in PCR reactions. The following genes are shown:
(c) EDG1; (d) PAK2; (e) Grb7; (f) TRAC-1.
EDG1
PAK2
Skeletal muscle
Brain
Heart
Colon
Thymus
Spleen
Kidney
Liver
Small intestine
Placenta
Lung
PBL

TRAC-1
Skeletal muscle
Brain
Heart
Colon
Thymus
Spleen
Kidney
Liver
Small intestine
Placenta
Lung
PBL
Grb7
Activation
PBMC
− +
CD8+
− +
CD4+
− +
CD19+
− +
CD14+
CD14+
CD14+
−
Brain
Placenta
PAK2 hit

Water
Water
Placenta
100 bp ladder
Water
CD14+
Placenta
100 bp ladder
Water
1 kb ladder
cDNA Panel
EDG1
β-actin
Activation
PBMC
− +
CD8+
− +
CD4+
− +
CD19+
− + −
cDNA panel
PAK2
β-actin
Activation
PBMC
− +
CD8+
− +

CD4+
− +
CD19+
− + −
Grb7
GAPDH
cDNA panel
Activation
PBMC
− +
CD8+
− +
CD4+
− +
CD19+
− + −
TRAC-1
GAPDH
cDNA Panel
(a) (b)
(c) (d)
(e) (f)
that prior culture and retroviral infection did not change the
basic properties of these primary T lymphocytes. Addition
of anti-CD28 in conjunction with anti-CD3 also led to high
IL-2 production from the GFP-negative population of cells
infected with CIG-LCK, -PLC␥1, -EDG1 and -PAK2 hits. The
GFP-positive population from these cells was, however, sig-
nificantly impaired in IL-2 production following anti-CD3
and anti-CD28 stimulation (Figure 6c). As expected, the

defect caused by the Lck, PLC␥1, EDG1 and PAK2 hits can
be completely rescued by stimulation using PMA and iono-
mycin (Figure 6c). Taken together, these results show that
Lck and PLC␥1 play a crucial role in IL-2 production from
primary T lymphocytes, consistent with their involvement
in membrane-proximal signaling events of T-cell activation.
More importantly, our results document for the first time
the involvement of the seven-transmembrane molecule
EDG1 and the serine/threonine kinase PAK2 in physiologi-
cal functions of T cells. Together, the results also demon-
strate a rapid system for further validating hits from
functional genetic screens using primary lymphocytes.
Discussion
In this article, we report a large-scale functional genetic
screen for inhibitors of TCR signaling. We isolated many
known signaling molecules - such as Lck, ZAP70, Syk,
PLC␥1 - as novel truncation mutants (probably created
during library preparation) with dominant-negative effects.
In addition, we also discovered molecules previously
unknown to this pathway, including transmembrane mole-
cules (EDG-1, IL-10R␣ and integrin ␣
2
), cytoplasmic
enzymes and adaptors (PAK-2, A-Raf-1, TCPTP, Grb7, SH2-B
and GG2-1), and cytoskeletal molecules (moesin and
vimentin; see Table 1). Of note, we also identified a novel
molecule, TRAC-1, which had lymphoid and hematopoietic
specific expression (Figure 5b).
We showed that EDG1, PAK2, and Grb7, genes originally
described in different contexts, are also expressed in lym-

phocytes (Figure 5). This is not unexpected, since the retro-
viral cDNA libraries were generated using mRNA from
Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. 21.11
Journal of Biology 2003, 2:21
No infection CRU5-GFP infection
Gate on
live cells
56%
25%
CD4
CD8
19%
R1
R1
GFP
84.1%
15.5%
Gate on live
lymphocytes
1000
800
600
400
200
0
FSC
SSC
10
0
10

1
10
2
10
3
10
4
10
0
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10

1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
1000
800
600

400
200
0
FSC
CD3
SSC
GFP
CIG-PLCγ1 hit
CIG-Lck hit
Geo mean = 87
Geo mean = 152
31.3% M1
CRU5-GFP
Geo mean = 3534
CRU5-IRES-GFP (CIG)
Geo mean = 322
100
80
60
40
20
0
120
Counts
250
200
150
100
50
0

Counts
200
160
120
80
40
0
200
160
120
80
40
0
32.4% M1
13.9% M1
15.0% M1
CIG-EDG1 hit
Geo mean = 124
CIG-PAK2 hit
Geo mean = 95
250
200
150
100
50
0
25.1% M1
200
160
120

80
40
0
17.1% M1
0
5
10
15
20
25
0
4,000
8,000
12,000
16,000
20,000
0
2
4
6
8
10
12
14
16
CIG Lck
hit
PLCγ
hit
EDG1

hit
PAK2
hit
GFP −
GFP +
IL2 (pg/ml)
CIG Lck
hit
PLCγ
hit
EDG1
hit
PAK2
hit
Anti-CD3 stimulated
Anti-CD3 + Anti-CD28 stimulated
PMA + ionomycin stimulated
Unstimulated
CIG Lck
hit
PLCγ
hit
EDG1
hit
PAK2
hit
CIG Lck
hit
PLCγ
hit

EDG1
hit
PAK2
hit
0
500
1,000
1,500
2,000
2,500
3,000
3,500
IL2 (pg/ml)
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10

3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10

3
10
4
10
0
10
1
10
2
10
3
10
4
(a)
(b)
(c)
Figure 6
The cDNA hits from the functional genetic screens inhibited activation
in human primary T lymphocytes. (a) Human PBL were cultured with
anti-CD3 and anti-CD28 for 3 days and then infected with the
retroviral CRU5-GFP vector, whereby GFP was expressed from the
constitutively active retroviral LTR promoter. Cells were stained with
anti-CD3-APC, or with anti-CD4-PE and anti-CD8-APC antibodies and
analyzed by FACS. The percentage of cells in each quadrant is shown.
(b) Human primary T lymphocytes were infected with vector alone
(CRU5-GFP and CRU5-IRES-GFP or CIG) or with the CIG vector
expressing the Lck, PLC␥1, EDG1 and PAK2 hits. The infection rate
was monitored by the percentage of GFP-positive cells (marked with
M1). The geometric mean of GFP for cells in marker M1 was shown
above the marker line. (c) Infected primary T lymphocytes were

allowed to rest and then sorted to give rise to GFP-negative (open
bars) and GFP-positive (filled bars) populations. Equal numbers of cells
were cultured without stimulation, with anti-CD3 or anti-CD3 plus
anti-CD28 antibodies, or with PMA plus ionomycin. Then, 40 h later
the culture supernatants were harvested and assayed for IL-2
production by ELISA. Note the difference in the scales and the standard
deviations with cells stimulated with anti-CD3 plus anti-CD28, or with
PMA plus ionomycin (lower panels) compared to the upper panels.
human lymphoid organs such as thymus, spleen, lymph
nodes and bone marrow. Our expression data are generally
consistent with those published by other investigators. For
example, EDG1 was reported to be expressed in human
natural killer cells [32] and dendritic cells [33]. PAK2 is
expressed ubiquitously in human tissues [20] and in Jurkat
cells [22]. Grb7 has a broad expression in human (pancreas,
placenta, kidney, prostate and small intestines) [34]. Grb7
was not easily detectable by northern blot in thymus, spleen
and PBL, but its expression was detected in specific lympho-
cyte subsets (Figure 5e). This indicates that our screen is
capable of identifying genes with potentially important roles
in lymphocyte activation whose expression is not limited to
the lymphoid system. The fact that these genes’ expression is
not limited to the lymphoid system does not diminish the
potential role they could play in lymphocyte activation. For
example, the Ras-Raf-MAP kinase pathway is ubiquitously
present in many tissues and cell types, as well as conserved
evolutionarily, but this pathway has also been shown to be
important in lymphocyte signaling.
In the ‘post-genomics’ era, the novelty of discovery lies in
assigning novel functions to gene products. In our screens,

for example, we identified two hits representing cytoplasmic
truncated versions of EDG-1, a receptor for S1P [16]. Inter-
estingly, FTY720, a potent immunosuppressant in advanced
clinical development, has been shown to act through
EDG-1 and S1P signaling pathways [35,36]. The fact that
truncated EDG-1 proteins were identified in our T-cell acti-
vation screen suggests potential intersections of the TCR sig-
naling pathway and the S1P signaling pathway, as well as
new insights into the mechanisms of action of FTY720.
Our results also call for attention to potential differences
between related family members. For example, PAK-1
(instead of PAK-2), c-Raf-1 (instead of A-Raf-1) and Grb2
(instead of Grb7) have been reported to be associated with
the TCR signal transduction pathway [25,37-40]. Our func-
tional genetic screens identified PAK2, A-Raf-1, and Grb7 as
important regulators of TCR-induced CD69 expression. It is
possible that the dominant-negative proteins we cloned also
inhibit other related family members. Alternatively, it is
equally possible that the previously reported dominant-
negative forms of PAK1, c-Raf-1, and to a lesser extent,
Grb2, may have inhibited PAK2, A-Raf-1 and Grb7, respec-
tively. In fact, binding of the human immunodeficiency
virus (HIV) Nef protein and subsequent activation of the
PAK-related kinase and phosphorylation of its substrate can
be readily detected in both infected primary T lymphocytes
and macrophages [41]. When the HIV-Nef-associated kinase
was characterized carefully, it became clear that this kinase
was PAK2 and not PAK1 [42,43]. This example supports the
notion that PAK2 could be the more relevant kinase in
T-cell signaling. Of course, it is entirely possible that these

related family members are not mutually exclusive in partic-
ipating in the TCR signal-transduction pathway.
In conclusion, we have demonstrated a successful approach
for discovering and validating, in a functionally relevant
context, important immune regulators on a genome-wide
scale. This approach provides a tool for functional cloning
of regulators in numerous signal-transduction pathways
[44,45]. For example, B-cell activation-induced CD69 expres-
sion [46] and, recently, the IL-4-induced immunoglobulin E
class switch [47], have also been shown to be amenable to
genetic perturbation following introduction of retroviral
cDNA or random cyclic peptide libraries. Importantly, the
outlined strategy, which requires no prior sequence informa-
tion of the players involved, does not bias the search to pre-
viously known signaling molecules, molecules flagged by
DNA-array technologies, or signaling molecules discovered
in other contexts. This approach has added to the list of
potential players in T-cell biology that have not been identi-
fied in other standard pathway-mapping techniques.
Materials and methods
Preparation of cDNA libraries
The mRNA extracted from human lymph nodes, thymus,
spleen and bone marrow was used to produce two ran-
domly primed cDNA libraries. For one library (-ATG)
inserts were directionally cloned and the second (+ATG)
non-directionally cloned and provided with three exoge-
nous ATGs in three frames. The resulting cDNAs were
cloned into the pTRA-exs vector [48] for doxycycline-
(Dox-) regulatable expression in cell lines expressing the
tetracycline transactivator protein (tTA) [9]. The total com-

bined complexity of the two pTRA-cDNA libraries was
5×10
7
independent clones.
Cell lines
Phoenix A cells were cultured in DMEM supplemented with
10% fetal calf serum (FCS), penicillin and streptomycin.
Human T-cell leukemia line Jurkat was obtained from Novar-
tis (Vienna, Austria) and was cultured in RPMI 1640 medium
supplemented with 10% FCS, penicillin and streptomycin.
Clone 4D9 with an optimal CD69 induction was obtained
after sequential FACS-sorting for low basal CD69 expression
and high induction of CD69 expression following TCR stim-
ulation. To produce the Jurkat-tTA cell line, Clone 4D9 was
infected with a reporter construct which expresses Lyt2
driven by a tetracycline responsive element (TRE) and a
retroviral construct, CtTA1H, which constitutively expresses
tTA [48]. The Jurkat-tTA cell clone 4D9#32 was obtained by
sorting for high Lyt2 expression in the absence of Dox and
low expression of Lyt2 in the presence Dox (10 ng/ml).
21.12 Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. />Journal of Biology 2003, 2:21
Transfection and infection
Phoenix A packaging cells were transfected with retroviral
vectors using calcium phosphate for 6 h following standard
protocols [49]. After 24 h, supernatant was replaced with
complete RPMI medium and virus was allowed to accumu-
late for 24 h at 32°C. Viral supernatant was collected, fil-
tered through a 0.2 ␮M filter and mixed with Jurkat cells or
human primary T lymphocytes at a density of 5 × 10
5

cells
per ml. Cells were spun at room temperature for 3 h
at 2,500 rpm, followed by overnight incubation at 37ºC.
Transfection and infection efficiencies were monitored
by FACS. Functional analysis was carried out at least 2 days
after infection.
Stimulation
For CD69 upregulation experiments, Jurkat cells were split
to 2.5 × 10
5
cells per ml 24 h prior to stimulation. Cells
were spun and resuspended at 5 × 10
5
cells per ml in fresh
complete RPMI medium in the presence of 300 ng/ml C305
(anti-Jurkat clonotypic TCR) hybridoma [2] supernatant,
100 ng/ml OKT3 (anti-CD3), 100 ng/ml SpvT3 (anti-CD3),
or PMA (5 ng/ml) for 20-26 h at 37ºC, and then assayed for
surface CD69 expression.
Antibodies and flow cytometry
Jurkat cells or human peripheral blood lymphocytes were
stained with FITC-conjugated monoclonal anti-mouse
CD8␣ (Lyt2), APC-conjugated mouse monoclonal anti-
human CD3, anti-human CD8, or anti-human CD69 anti-
bodies, and PE-conjugated mouse monoclonal anti-human
CD3 or anti-CD4 antibodies (all from Caltag, Burlingame,
USA) at 4ºC for 20 min and analyzed using a FACSCalibur
instrument (Becton Dickinson, Franklin Lakes, USA) with
the CellQuest software. Fluorescent-activated cell sortings
were performed on the MoFlo instruments (Cytomation,

Fort Collins, USA).
Genetic screens
Phoenix A packaging cells were transfected with pTRA-
cDNA libraries. Supernatant containing packaged viral parti-
cles was used to infect 3.5 × 10
8
Jurkat-tTA cells with an
efficiency of 52% based on parallel infection with TRA-GFP
[48]. After 4 days of cDNA expression, library-infected
cells were stimulated with 300 ng/ml C305 for 20-30 h,
stained with APC-conjugated anti-CD69 and PE-conju-
gated anti-CD3, and 1% of total cells with the desired
CD69
low
CD3
+
phenotype were isolated using MoFlo.
Sorting was repeated multiple times with a 6-day rest period
between stimulations until the population was significantly
enriched for the desired CD69
low
CD3
+
phenotype. Single
cells were deposited to 96-well plates and expanded in the
presence and absence of Dox, stimulated and analyzed for
CD69 upregulation.
Isolation of cDNA inserts
PCR primers were designed to specifically amplify the
inserts from pTRA-cDNA libraries. The primers contained

flanking BstXI sites for subsequent cloning to the pTRA-
IRES-GFP and CRU5-IRES-GFP vectors [48,50]. BstXTRA5G:
5؅-TTGCAGAACCACCACCTTGGGCTCTTAACCTAGGCCGA-
TC-3؅. BstXTRA3D: 5؅-TTGCAGAACCAATTTAATGGCGGC-
CAGTCAGGCCATCGTCG-3؅. RT-PCR cloning was achieved
with kits from Clontech (Palo Alto, USA) or Life Technolo-
gies (Carlsbad, USA). The gel-purified RT-PCR fragments
were sequenced as well as digested with BstXI for sub-
cloning into the retroviral pTRA-IRES-GFP or CRU5-IRES-
GFP vectors.
Semi-quantitative PCR analysis
Human Blood Fractions MTC panel (Clontech) with normal-
ized, first-strand cDNA preparations from RNA of various
purified cells were used as templates. CD19
+
cells were acti-
vated with 2 ␮l/ml pokeweed mitogen for 4 days, mononu-
clear cells with 2 ␮l/ml pokeweed mitogen and 5 ␮g/ml con-
canavalin A for 3 days, CD4
+
cells with 5 ␮g/ml concanavalin
A for 3-4 days, and CD8
+
cells with 5 ␮g/ml phytohemagglu-
tinin for 3 days. The following primers were used to amplify
various cDNA fragments: EDG1: forward primer 5؅-GCAA-
GAACATTTCCAAGGCCAGCC-3؅, reverse primer 5؅-GGGT-
GTGGGATGTACAGGGCATCC-3؅, 35 cycles; PAK2: forward
primer 5؅-CGGAGAACTGGAAGATAAGCCTCC-3؅, reverse
primer 5؅-AAAGCCAACATGGATGGTGTGCTC-3؅, 35 cycles;

Grb7: forward primer 5؅-ATGCCCACTGACTTCGGTTT-3؅,
reverse primer 5؅-GATCCGAAGCCCCTTGTGT-3؅, 40 cycles;
TRAC-1: forward primer 5؅-TTACACCAGCCTGTCCGGA-3؅,
reverse primer 5؅-CAGACTGGTAGCAATACAGGAACG-3؅,
35 cycles.
Commercially available primers were used for GAPDH
(PerkinElmer, Wellesley, USA) and ␤-actin (Clontech), 25
cycles. The PCR products were then electrophoresed on
agarose/ethidium bromide gels.
Northern blot analysis
Human Multiple Tissue Northern Blots were purchased
from Clontech. The following probes were used: EDG1,
base pairs 1-1,023 of its open reading frame; PAK2, base
pairs 1-341 of its open reading frame; Grb7, base pairs
1,268-1,599 of its open reading frame; and TRAC-1, base
pairs 1-509 of its open reading frame.
Culture and infection of primary T lymphocytes
Commercially available primary blood mononuclear cells
(PBMC; AllCells LLC, Berkeley, USA) were cultured in RPMI
+ 10% FCS for 1-2 h in tissue culture flasks to allow
macrophages and other adhering cells to settle down. The
suspended cells were cultured with anti-CD3 (30 ng/ml)
Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. 21.13
Journal of Biology 2003, 2:21
and anti-CD28 (100 ng/ml) for 2 days to allow T cells to
expand and other cell types to gradually die off. These
primary T lymphocytes were infected with 1 ml retroviral
supernatant in a 24-well plate. One day after infection, 1 ml
spent medium from the bulk culture was added to each
well. The cells were further expanded for a few days with

addition of fresh RPMI + 10% FCS. Such an expansion also
allowed the cells to return to the resting state with low
CD69, CD25, and CD40L expression. Cells were then sorted
by FACS, on the basis of GFP expression, directly into a
round bottom 96-well plate coated with anti-CD3 alone,
anti-CD3 + anti-CD28, or not coated. To the uncoated
wells, PMA (5 ng/ml final) and ionomycin (1 ␮M final)
were added. Then, 40 h later, supernatants were harvested
for IL-2 measurement using commercial reagents (R&D
Systems, Minneapolis, USA).
Additional data files
The following are provided as additional materials with
this article online: details of the selection and infection of
Jurkat clone 4D9 (Additional data file 1); construction of
the pTRA-cDNA libraries and assessing the efficiency of
infection (Additional data file 2); distinction between CD3
-
,
CD3
low
and CD3
high
cell populations (Additional data file 3);
distribution of Dox ratios among the 2,828 single-cell
clones analyzed (Additional data file 4); details of clones
with TCR␤ hits (Additional data file 5); a summary of the
genetic screen for inhibitors of TCR-induced CD69 expres-
sion (Additional data file 6); characterization of additional
hits from the T-cell activation screen (Additional data file
7); correlation of the CD69 inhibitory phenotype with the

cDNA expression level (Additional data file 8).
Acknowledgements
We thank Arthur Weiss (UCSF) for providing the C305 hybridoma and
ZAP70 constructs used for positive controls, our collaborators Ulf
Korthaeuer, Max Woisetschlager, Christoph Heusser, Jutta Heim and
N. Rao Movva from Novartis for their support during this work. We
thank Jim Lorens, Anup Nagin, Sacha Holland, Xian Wu, Monette Aujay,
and Mel Fox for their excellent technical advice and assistance, Jianing
Huang and Garry Nolan for critical reading of the manuscript, and Louis
Tamayo, Carine Richards, and Amelia Cervantes for assistance with the
preparation of the manuscript.
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21.16 Journal of Biology 2003, Volume 2, Issue 3, Article 21 Chu et al. />Journal of Biology 2003, 2:21